Waitlisting and liver transplantation for MetALD in the United States: An analysis of the UNOS national registry.

BACKGROUND AND AIMS: The new steatotic liver disease (SLD) nomenclature introduced metabolic and alcohol-associated liver disease (MetALD), describing the intersection of metabolic dysfunction-associated steatotic liver disease and alcohol-associated liver disease. Waitlisting and liver transplantation for MetALD are not well defined. We aimed to develop and validate an algorithm for identifying SLD phenotypes and assessing trends in waitlisting and transplant outcomes. APPROACH AND RESULTS: We conducted a retrospective cohort study using the United Network for Organ Sharing registry, supplemented with detailed single-center data. We developed 5 candidate algorithms for SLD classification and calculated their diagnostic performance. Trends in waitlist registrations and transplants were estimated, and competing risk analyses and Cox regression models were conducted to assess waitlist removal and posttransplant outcomes among SLD phenotypes. The best-performing algorithm demonstrated substantial agreement (weighted kappa, 0.62) for SLD phenotypes, with acceptable sensitivity (73%) for MetALD. Between 2002 and 2022, waitlist registrations and transplants for MetALD increased 2.9-fold and 3.3-fold, respectively. Since 2013, there has been a significant increase in the absolute number of waitlist registrations (122 per year; 95% CI, 111-133) and transplants (107 per year; 95% CI, 94-120) for MetALD. Patients with MetALD experienced higher waitlist removal (adjusted subdistribution hazard ratio, 1.10; 95% CI, 1.03-1.17), all-cause mortality (adjusted hazard ratio, 1.13; 95% CI, 1.03-1.23), and graft failure (adjusted hazard ratio, 1.12; 95% CI, 1.03-1.21) than those with alcohol-associated liver disease. CONCLUSIONS: We developed and validated an algorithm for identifying SLD phenotypes in UNOS. MetALD is the third leading etiology among those waitlisted and underwent transplantation, exhibiting worse pretransplantation and posttransplantation outcomes compared to alcohol-associated liver disease. Identifying and addressing factors determining poor outcomes is crucial in this patient population.

Association of psychosocial risk factors and liver transplant evaluation outcomes in metabolic dysfunction-associated steatotic liver disease.

The Stanford Integrated Psychosocial Assessment for Transplantation (SIPAT) is a standardized psychosocial assessment tool used in liver transplantation (LT) evaluation and has been primarily studied in patients with alcohol-associated liver disease. We aimed to evaluate the relationship between SIPAT score and metabolic syndrome severity and LT waitlist outcomes in a large cohort of patients with metabolic dysfunction-associated steatotic liver disease (MASLD). We performed a single-center retrospective cohort study of patients with MASLD evaluated for LT from 2014 to 2021. The utility of the previously defined total SIPAT cutoff (<21 [excellent/good candidates] vs. ≥21 [minimally acceptable/high-risk candidates]) was studied. Multivariable logistic regression analyses examined associations between continuous SIPAT scores and LT waitlisting outcomes. The Youden J statistic was used to identify the optimal SIPAT cutoff for patients with MASLD. A total of 480 patients evaluated for transplant with MASLD were included. Only 9.4% of patients had a SIPAT score ≥21. Patients with SIPAT score ≥21 had higher hemoglobin A1c compared to patients with lower psychosocial risk (median [IQR]: 7.8 [6.0-9.7] vs. 6.6 [5.8-7.9]; p = 0.04). There were no other differences in metabolic comorbidities between SIPAT groups. Increasing SIPAT score was associated with decreased odds of listing (OR: 0.82 per 5-point increase; p = 0.003) in multivariable models. A SIPAT of ≥12 was identified as the optimal cutoff in this population, resulting in an adjusted OR for a listing of 0.53 versus SIPAT <12 ( p = 0.001). In this large cohort of patients with MASLD evaluated for LT, few patients met the previously defined high SIPAT cutoff for transplant suitability. Nevertheless, increasing the SIPAT score was associated with waitlist outcomes. Our suggested SIPAT cutoff of ≥12 for patients with MASLD warrants further external validation using data from other centers.

Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli.

The methylation of unactivated carbon and phosphorus centers is a burgeoning area of biological chemistry, especially given that such reactions constitute key steps in the biosynthesis of numerous enzyme cofactors, antibiotics, and other natural products of clinical value. These kinetically challenging reactions are catalyzed exclusively by enzymes in the radical S-adenosylmethionine (SAM) superfamily and have been grouped into four classes (A-D). Class B radical SAM (RS) methylases require a cobalamin cofactor in addition to the [4Fe-4S] cluster that is characteristic of RS enzymes. However, their poor solubility upon overexpression and their generally poor turnover has hampered detailed in vitro studies of these enzymes. It has been suggested that improper folding, possibly caused by insufficient cobalamin during their overproduction in Escherichia coli, leads to formation of inclusion bodies. Herein, we report our efforts to improve the overproduction of class B RS methylases in a soluble form by engineering a strain of E. coli to take in more cobalamin. We cloned five genes ( btuC, btuE, btuD, btuF, and btuB) that encode proteins that are responsible for cobalamin uptake and transport in E. coli and co-expressed these genes with those that encode TsrM, Fom3, PhpK, and ThnK, four class B RS methylases that suffer from poor solubility during overproduction. This strategy markedly enhances the uptake of cobalamin into the cytoplasm and improves the solubility of the target enzymes significantly.

Stereochemical and Mechanistic Investigation of the Reaction Catalyzed by Fom3 from Streptomyces fradiae, a Cobalamin-Dependent Radical S-Adenosylmethionine Methylase.

Fom3, a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase, has recently been shown to catalyze the methylation of carbon 2″ of cytidylyl-2-hydroxyethylphosphonate (HEP-CMP) to form cytidylyl-2-hydroxypropylphosphonate (HPP-CMP) during the biosynthesis of fosfomycin, a broad-spectrum antibiotic. It has been hypothesized that a 5'-deoxyadenosyl 5'-radical (5'-dA•) generated from the reductive cleavage of SAM abstracts a hydrogen atom from HEP-CMP to prime the substrate for addition of a methyl group from methylcobalamin (MeCbl); however, the mechanistic details of this reaction remain elusive. Moreover, it has been reported that Fom3 catalyzes the methylation of HEP-CMP to give a mixture of the ( S)-HPP and ( R)-HPP stereoisomers, which is rare for an enzyme-catalyzed reaction. Herein, we describe a detailed biochemical investigation of a Fom3 that is purified with 1 equiv of its cobalamin cofactor bound, which is almost exclusively in the form of MeCbl. Electron paramagnetic resonance and Mössbauer spectroscopies confirm that Fom3 contains one [4Fe-4S] cluster. Using deuterated enantiomers of HEP-CMP, we demonstrate that the 5'-dA• generated by Fom3 abstracts the C2″- pro-R hydrogen of HEP-CMP and that methyl addition takes place with inversion of configuration to yield solely ( S)-HPP-CMP. Fom3 also sluggishly converts cytidylyl-ethylphosphonate to the corresponding methylated product but more readily acts on cytidylyl-2-fluoroethylphosphonate, which exhibits a lower C2″ homolytic bond-dissociation energy. Our studies suggest a mechanism in which the substrate C2″ radical, generated upon hydrogen atom abstraction by the 5'-dA•, directly attacks MeCbl to transfer a methyl radical (CH3•) rather than a methyl cation (CH3+), directly forming cob(II)alamin in the process.

Comprehensive Review of Cardiovascular Disease Risk in Nonalcoholic Fatty Liver Disease.

Nonalcoholic Fatty Liver Disease (NAFLD) is a growing global phenomenon, and its damaging effects in terms of cardiovascular disease (CVD) risk are becoming more apparent. NAFLD is estimated to affect around one quarter of the world population and is often comorbid with other metabolic disorders including diabetes mellitus, hypertension, coronary artery disease, and metabolic syndrome. In this review, we examine the current evidence describing the many ways that NAFLD itself increases CVD risk. We also discuss the emerging and complex biochemical relationship between NAFLD and its common comorbid conditions, and how they coalesce to increase CVD risk. With NAFLD's rising prevalence and deleterious effects on the cardiovascular system, a complete understanding of the disease must be undertaken, as well as effective strategies to prevent and treat its common comorbid conditions.

TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases.

Cobalamin-dependent radical S-adenosylmethionine (SAM) methylases play vital roles in the de novo biosynthesis of many antibiotics, cofactors, and other important natural products, yet remain an understudied subclass of radical SAM enzymes. In addition to a [4Fe-4S] cluster that is ligated by three cysteine residues, these enzymes also contain an N-terminal cobalamin-binding domain. In vitro studies of these enzymes have been severely limited because many are insoluble or sparingly soluble upon their overproduction in Escherichia coli. This solubility issue has led a number of groups either to purify the protein from inclusion bodies or to purify soluble protein that often lacks proper cofactor incorporation. Herein, we use TsrM as a model to describe methods that we have used to generate soluble protein that is purified in an active form with both cobalamin and [4Fe-4S] cluster cofactors bound. Additionally, we highlight the methods that we developed to characterize the enzyme following purification.